Dimitrov Antony S, Rawat Satinder S, Jiang Shibo, Blumenthal Robert
Laboratory of Experimental and Computational Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, Maryland 21702-1201, USA.
Biochemistry. 2003 Dec 9;42(48):14150-8. doi: 10.1021/bi035154g.
The N-terminal fusion peptide and the interfacial sequence preceding the transmembrane anchor of HIV-1 gp41 are required for viral fusion. Studies with synthetic peptides indicated that these regions function by destabilizing membranes, which is regarded as a crucial step in the membrane fusion reaction. However, it is not clear whether membrane destabilization is induced by these sequences in the intact gp41. We address this question by examining fusion and destabilization of membranes expressing HIV-1(IIIB) wild-type Env and two mutant Envs. (1) A Glu residue at position 2 of the gp41 fusion peptide is substituted for Val (V2E) to produce one mutant. (2) Residues 665-682 in the membrane-proximal domain are deleted to form the other. The process of membrane destabilization was monitored by the influx of Sytox, an impermeant fluorescent dye, into the Env-expressing cells following the interaction with CD4-CXCR4 complexes, and fusion was monitored by observing dye transfer between Env-expressing cells and appropriate target cells. We also monitored the conformational changes in the Envs following their interactions with CD4 and CXCR4 by immunofluorescence using an anti-gp41 mAb that reacts with the six-helix bundle. In contrast to the wild type, both Env mutants did not mediate cell fusion. The V2E Env did not mediate membrane destabilization. However, the Env with an unmodified fusion peptide but with a deletion of residues 665-682 in the membrane-proximal domain did mediate membrane destabilization. The wild type and both mutant Envs undergo conformational changes detected by the anti-gp41 six-helix bundle mAbs. Our results suggest that in intact HIV-1 Env the membrane-proximal domain is not required for membrane perturbations, but rather enables the bending of gp41 that is required for viral and target membranes to come together. Moreover, the observation that the Delta665-683 Env self-inserts its fusion peptide but does not cause fusion suggests that self-insertion of the fusion peptide is not sufficient for HIV-1 Env-mediated fusion.
HIV-1 gp41的N端融合肽和跨膜锚定之前的界面序列是病毒融合所必需的。对合成肽的研究表明，这些区域通过使膜不稳定发挥作用，这被认为是膜融合反应中的关键步骤。然而，尚不清楚在完整的gp41中这些序列是否会诱导膜不稳定。我们通过检测表达HIV-1（IIIB）野生型Env和两种突变型Env的膜的融合和不稳定来解决这个问题。（1）将gp41融合肽第2位的Glu残基替换为Val（V2E）以产生一种突变体。（2）缺失膜近端结构域中的665-682位残基以形成另一种突变体。通过一种非渗透性荧光染料Sytox流入与CD4-CXCR4复合物相互作用后的Env表达细胞来监测膜不稳定过程，并且通过观察Env表达细胞与合适靶细胞之间的染料转移来监测融合。我们还使用与六螺旋束反应的抗gp41单克隆抗体通过免疫荧光监测Env与CD4和CXCR4相互作用后的构象变化。与野生型相反，两种Env突变体均不介导细胞融合。V2E Env不介导膜不稳定。然而，具有未修饰融合肽但膜近端结构域缺失665-至682位残基的Env确实介导了膜不稳定。野生型和两种突变型Env均经历了抗gp41六螺旋束单克隆抗体检测到的构象变化。我们的结果表明，在完整的HIV-1 Env中，膜近端结构域不是膜扰动所必需的，而是使gp41弯曲从而使病毒膜和靶膜聚集在一起。此外，Delta665-683 Env自我插入其融合肽但不引起融合这一观察结果表明，融合肽的自我插入不足以实现HIV-1 Env介导的融合。
